The Role And Molecular Mechanism Of Autophahgy In Delphinidin-induced Anticancer Effects For HER-2 Positive Breast Cancer

Our preliminary study have demonstrated Black Rice Anthocyanidin obiviously suppresses cell growth and metastasis of HER-2 positive breast cancer,while exerts little cytotoxicity in nomal breast cancer and HER-2 negative breast cancer.As the main monomer in anthocyanidin,it is still unknown on the anticancer effect and mechanism of delphinidin in HER-2 positive breast cancer.Autophagy and apoptosis are two processes in mediation cell death.Autophagy plays a double-edge sword effect,which means autophagy can protect cell from apoptotic cell death or promoted autophagy-related cell death under stress metabolism microenviroment.Some studies have shown that delphinidin could induce protective autophagy in mouse fibroblast cells;under starved condition,induced autophagy can promote cancer cells survival through maintaining circulation of nitrogen and decrease the life-span of patiens with breast caner;in vitro 3D experiment,metastatic cancer cells hold survival via autophagy way.It is reported that m TOR and AMPK signaling pathway are classical pathways in autophagic regulation,and related to cell growth and energy metabolism.The active AMPK sensor and suppressive m TOR receptors could induce protective autophagy in breast cancer cells under low oxygen respons.Meanwhile,autophagy can be induced through endoplasmic reticulum stress.Consequently,it is necessary to understand the relationshilp between autophagy and anticancer effect of delphinidin,and the mechanism of autophagy induced by delphinidin.Objective Base on preliminary study of our group,it is aim to explore the inhibition effect of delphinidin in HER-2 positive breast cancer,and to detect the role of delphinidin-induced autophagy in the inhibition effect.Our study illuminated the effect of delphinidin against HER-2 positive breast cancer,and provided a new preventative and therapeutic measure from the view of autophagy for HER-2 positive breast cancer.HER-2(human epidermal growth factor receptor-2,HER-2)positive MDA-MB-453 breast cancer cells were treated by delphinidin in different concentration(1)Proliferation of MDA-MB-453 cells was detected by CCK-8 assay after 48 h.(2)TUNEL assay and Western blot were used to explore apoptotic status.TUNEL assay was used to count the number of apoptotic cells;Western blot was used to analysis activity of protein caspase-3 and caspase-9 mediated apoptosis.(3)The assays of autophagy identification include autophagosomes observation by transmission electron microscope,expression of transformed LC3-II by fluorescence punctate dot assay,immunofluorescence and Western blot,and expression of Atg5-Atg12,the protein of mediation autophagosome membrane,by Western blot.(4)Treatment of 3-MA(5 mmol/L)with delphinidin(80 mmol/L)was used to detect the effect of autophagy inhibition.Fluorescence punctate dot assay and Western blot were used to identify autophagy inhibition status;CCK-8 and TUNEL assay were used to analyze proliferation and apoptosis.(5)Immunoblot was used to investigate mechanism of autophagy,including expression of proteins in m TOR signaling pathway(AKT/m TOR/p70s6k/e IF4e)and AMPK signaling pathway(LKB1/AMPK/FOXO3a/ ULK1).Materials and MethodsResults(1)Delphinidin inhibited proliferation of MDA-MB-453 cells,increased the number of TUNEL positive cells and the expression of cleaved caspase-3/9.(2)Delphinidin increased the number of GFP-LC3 punctate dots,accumulation of protein LC-3,formation of autophagosome,transformation of LC3-I/II and expression of autophagy related protein of Atg5-Atg12.(3)Autophagy was induced by delphinidin through inhibition of m TOR signaling pathway with downregulating phosphorylation of AKT,m TOR,e IF4 E and p70s6 k,and through activation of AMPK signaling pathway by upregulated activity of LKB1,AMPK,FOXO3 a and ULK1 in MDA-MB-453 cells.(4)Autophagy suppression boosted delphinidin-induced apoptotic cell death and antiproliferative effeciency of MDA-MB-453 cells.Conclusion Delphinidin promoted proliferation inhibition and caspase-3/9 dependent apoptosis and autophagy in HER-2 positive MDA-MB-453 breast cancer cells.Autophagy inhibition could enhance the anticancer effect of delphinidin in MDA-MB-453 cells.Delphinidin induced protective autophagy and facilitate cell survival via surpression m TOR signaling pathway and activation AMPK signaling pathway in MDA-MB-453 cells.